The long term objective are as follows. (1) Develop a new methodology for synthesizing RNA. Although considerable progress has been achieved over the past twenty years, developing mutually orthogonal protecting groups remains a major unsolved barrier to the chemical synthesis of RNA in high yield and purity. The proposed approach involves using phosphoramidite chemistry to synthesize RNA in a 3' to 5' direction on supports. The route will test synthons having acid labile 2' - protection, base labile groups on the purines/pyrimidines and that link the growing RNA to the support, and 5' -blocking groups (eliminated after each addition) that are removed by strong nucleophiles or fluoride anion. RNA prepared by the chemistry should provide a mechanism for introducing reporter groups, probes, and modified nucleotides into RNA. (2) Improve DNA synthesis methods. Synthetic DNA, as prepared by current procedures, meets most of the requirements of biochemists and molecular. However for applications requiring cloned, synthetic DNA, the modifications per base (approx. 1.3%) are excessive. In order to improve DNA quality, a new synthesis strategy is proposed that eliminates a major source of these modifications (the acid detritylation step) and should significantly reduce the mutations found in cloned, synthetic DNA. (3) Synthesize and biochemically study new DNA and RNA analogs. A new DNA derivative having sulfur in place of oxygen at the two nonbridging valencies (dithioate DNA) was synthesized and shown to be nuclease resistant, a potent inhibitor of retroviruses, and potentially an antisense oligonucleotide as it stimulates RNase H. Proposed additional studies include testing this analog as a probe for nucleic acid structure, using it to study polymerase and other enzymes, and as a hapten for generating catalytic antibodies with phosphodiesterase activity. Extension to dithioate RNA is proposed primarily to prepare nuclease resistant ribozymes - a potential new class of therapeutic drugs. Two additional series of analogs that will be synthesized and studied biochemically are the 3'/5' methylene phosphonate and 3'/5' phosphorothioate linked DNA. Initially these derivatives will be characterized relative to nuclease resistance, ability to stimulate RNase H, and for stability with complementary oligomers. The most promising derivatives will be studied as potential antisense oligonucleotides against viruses, other diseases, and specific cellular genes. Phosphorothioate bridged oligomers are also expected to be useful probes for studying mechanisms of phosphorolysis by enzymes and ribozymes. Another useful DNA analog, which will be synthesized and studied biochemically for the first time, is diamidate DNA. It may be especially useful for inhibiting translation and RNA splicing through hybridization arrest. Another potentially exciting use will be its ability to form triplexex and thus inhibit gene expression at the DNA level. All these analogs are uncharacterized and, in most cases, they have not been synthesized previously. Thus considerable basic biochemistry must be carried out to assess their potential in basic research and as therapeutic drugs.